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EP1689853B1 - Separation de spermatozoides par electrophorese - Google Patents

Separation de spermatozoides par electrophorese Download PDF

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Publication number
EP1689853B1
EP1689853B1 EP04761402A EP04761402A EP1689853B1 EP 1689853 B1 EP1689853 B1 EP 1689853B1 EP 04761402 A EP04761402 A EP 04761402A EP 04761402 A EP04761402 A EP 04761402A EP 1689853 B1 EP1689853 B1 EP 1689853B1
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EP
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Prior art keywords
sperm
process according
electrophoresis
ion
chamber
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German (de)
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EP1689853A4 (fr
EP1689853A1 (fr
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Robert John Aitken
Christopher James Ainsworth
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The University of Newcastle
Newcastle Innovation Ltd
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The University of Newcastle
Newcastle Innovation Ltd
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Priority claimed from AU2003905474A external-priority patent/AU2003905474A0/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0608—Germ cells
    • C12N5/0612—Germ cells sorting of gametes, e.g. according to sex or motility
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D57/00—Separation, other than separation of solids, not fully covered by a single other group or subclass, e.g. B03C
    • B01D57/02—Separation, other than separation of solids, not fully covered by a single other group or subclass, e.g. B03C by electrophoresis
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D71/00—Semi-permeable membranes for separation processes or apparatus characterised by the material; Manufacturing processes specially adapted therefor
    • B01D71/06—Organic material
    • B01D71/50—Polycarbonates

Definitions

  • the present invention relates to the use of membrane-based electrophoresis technology for isolating/enriching sperm population(s) from semen.
  • electrophoresis used up to now is free-flow electrophoresis. This form involves laminar flow of cells through a specially designed chamber within an electric field. The different mobilities (different charge to mass ratios) of the cells in the electric field allows the cells to separate and they are collected through multiple channels at the end of the chamber. The extent to which these correlate with functional properties of the cells is variable.
  • Cell electrophoresis is a high resolution separation method.
  • traditional electrophoresis sub-populations of cells for which no affinity ligand has been developed and for which there is no distinct size or density range are often separable on the basis of their electrophoretic mobility, which may be related to their function.
  • the electrophoretic mobility of a cell is directly correlated with the cellular negative surface charge density.
  • the surface charge on cells will vary depending on the cell type, relative freshness of the cells, and the pH of the electrophoresis buffer used for separation. At physiological pH (around neutral), cells have a net negative surface charge and when placed in an electric field, they are deflected or moved towards the anode.
  • the present inventors provide a reliable, reproducible, rapid, efficient, and cost-effective method of enriching a sperm population from semen or other solutions using membrane-based electrophoresis technology.
  • the present invention provides a process for separating a sperm type from a sperm population by electrophoresis as defined in claim 1 of the claims attached hereto.
  • the sperm type can have a desired characteristic and that type is-separated from the sperm population.
  • a desired characteristic include, but not limited to, motility, robustness, gender, genetic make-up, morphological normality, fertilizing potential, and combination thereof, preferably, the sperm type has fertilizing potential.
  • the sperm type can have a undesired characteristic and that type is removed from the sperm population.
  • An undesired characteristic would include poor motility, poor morphology, high levels of DNA damage and high levels of reactive oxygen species generation.
  • leukocytes are also removed from the sperm preparations used for assisted conception, as these cells are potentially damaging to sperm.
  • the process comprises:
  • the sperm sample preferably contains at least two populations of sperm.
  • the electric potential can be applied until at least one sperm type reaches a desired purity.
  • one or more ion-permeable barriers are electrophoresis membranes having a characteristic average pore size and pore size distribution.
  • all of the ion-permeable barriers are membranes have a characteristics average pore size and pore size distribution.
  • the first ion-permeable barrier is a large pore sized membrane.
  • suitable large pore sized membranes include, but are not limited to polycarbonate or polyacrylamide.
  • a large pore size membrane has a pore size of about 1 to about 10 ⁇ m.
  • the membrane has a pore size of about 5 ⁇ m.
  • electrophoresis is carried out with a voltage range from about 1 to 200 V.
  • Electrophoresis can be carried out with an applied current of up to about 75 mA.
  • Electrophoresis can be carried out with a field strength of 1 to 100 V/cm.
  • the present inventors have found that a field strength from about 16 to 20 V/cm is particularly suitable.
  • Electrophoresis is carried out from several seconds to about 15 minutes in an apparatus having a sample volume of about 100 ⁇ l to about 4 ml.
  • the sperm can diluted in buffer having a concentration of between about 1 to 100 mM.
  • sperm concentrations of between about 0.1 and 250 x 10 6 /ml can be readily processed.sperm concentrations of between 15 x 10 6 /ml and 140 x 10 6 /ml have been processed successfully.
  • the step of applying an electric potential between the electrodes is maintained until at least one sperm type reaches a desired purity level in the first or second sample chamber.
  • the electrophoresis separation membranes are preferably made from polyacrylamide and have a molecular mass cut-off of at least about 5 kDa.
  • the molecular mass cut-off of the membrane will depend on the sample being processed, the other molecules in the sample mixture, and the type of separation carried out.
  • the first ion-permeable barrier is a large pore sized membrane such as a polycarbonate membrane.
  • the second and third barriers are preferably restriction membranes having a molecular mass cut off less than that of the first membrane.
  • a restriction membrane is also preferably formed from polyacrylamide. The molecular mass cut-off of the restriction membranes will depend on the sample being processed, the other molecules in the sample mixture, and the type of separation carried out. It will be appreciated that the second ion-permeable barrier may have a different molecular mass cut off to the third ion-permeable barrier.
  • the second and third ion-permeable barriers are membranes having characteristic average pore size and pore-size distribution.
  • the present invention can result in recovery rates of at least 50% active sperm type of choice.
  • the recovery rates can be much higher and in the order of about 60% or greater.
  • recovery rates are at least about 70%, more preferably at least about 80%, even more preferably at least about 90%, or up to about 95% of the at least one sperm type remains viable or substantially unchanged after separation.
  • the sample may be processed in a static form in batches or may be processed by moving the sample and electrolyte in streams through the apparatus.
  • the first sample chamber is called stream 1 and the second sample chamber is called stream 2.
  • Preferred buffer concentrations are between about 1 to 100 mM.
  • Suitable buffer or electrolyte can be used.
  • Suitable buffers include, but not limited to, sperm-compatibly biological buffers and components such as HEPPS, HEPES, BisTris, sodium chloride, phosphate buffer salts, sucrose, glucose and mannitol. In experiments outlined below, a 10 mM buffer of HEPES, 30 mM NaCl and 0.2 M sucrose has been found to be particularly useful. It will be appreciated, however, that any other suitable buffer can be used.
  • the present invention provides use of a membrane-based electrophoresis apparatus to separate a sperm type from a sperm population.
  • the process according to the present invention is suitable for separating sperm from animals including humans.
  • the separated sperm can be used for artificial insemination (Al) or in vitro fertilisation (IVF) applications.
  • the separated sperm can be stored by freezing for example, or used fresh, depending on the application.
  • Gradiflow TM and Microflow TM are trademarks owned by Gradipore Limited, Australia.
  • ion-permeable barriers that substantially prevent convective mixing between the adjacent chambers of the apparatus or unit are placed in an electric field and a cell type or population in the sample is selectively transported through an ion-permeable barrier.
  • the particular ion-permeable barriers used will vary for different applications and generally have characteristic average pore sizes and pore size distributions and/or isoelectric points allowing or substantially preventing passage of different components.
  • the apparatus typically includes a separation cartridge or zone which houses a number of membranes forming at least two chambers, cathode and anode in respective electrode chambers connected to a suitable power supply, reservoirs for samples, buffers and electrolytes, pumps for passing samples, buffers and electrolytes, and cooling means to maintain samples, buffers and electrolytes at a required temperature during electrophoresis.
  • the cartridge or zone contains at least three substantially planar membranes disposed and spaced relative to each other to form two chambers through which sample or solvent can be passed.
  • a separation membrane is disposed between two outer membranes (termed restriction membranes as their molecular mass cut-offs are usually smaller than the cut-off of the separation membrane).
  • restriction membranes are located adjacent to an electrode.
  • the cartridge is described in AU 738361 .
  • Description of membrane-based electrophoresis can be found in US 5039386 and US 5650055 in the name of Gradipore Limited, incorporated herein by reference.
  • An apparatus particularly suitable for use in isoelectric separation applications can be found in WO 02/24314 in the name of The Texas A&M University System and Gradipore Limited.
  • One electrophoresis apparatus suitable for use in the present invention comprises:
  • the apparatus may further comprise one or more of:
  • the ion-permeable barriers typically prevent substantial convective mixing of contents of the adjacent chambers.
  • the first ion-permeable barrier is a membrane having a characteristic average pore size and pore size distribution. In one preferred form, all the ion-permeable barriers are membranes having a characteristic average pore size and pore size distribution. This configuration of the apparatus is suitable for separating compounds on the basis of charge and or size.
  • the second and third ion-permeable barriers are membranes having a characteristic average pore size and pore-size distribution.
  • an inducible electro-endo-osmotic membrane can be used in at least one of the second or third ion-permeable barriers.
  • the inducible electro-endo-osmotic membrane is preferably a cellulose tri-acetate (CTA) membrane.
  • CTA cellulose tri-acetate
  • the inducible electro-endo-osmotic membrane can be formed from any other suitable membrane material such as poly(vinyl alcohol) cross-linked with glutaraldehyde (PVAI+glut).
  • a polycarbonate membrane having a pore size of about 5 ⁇ m is particularly suitable for use in the apparatus. It will be appreciated that other molecular mass cut-offs would also be suitable for the apparatus.
  • the pore size should be sufficiently large for sperm to pass.
  • the electrophoresis apparatus may contain a separation unit housing the chambers and ion-permeable barriers which is provided as a cartridge or cassette fluidly connected to the electrolyte reservoir(s) and, if present, the sample reservoirs.
  • a sample containing cells to be separated is placed in the first or second sample chamber.
  • Electrolyte is placed in the first and second electrolyte chambers.
  • Electrolyte or other liquid can be placed in the first and/or second sample chamber.
  • An electric potential is applied to the electrodes wherein a cell type in the first and/or second sample chamber is caused to move through a diffusion barrier to the second and/or first sample chamber.
  • An example of an application for the present invention has been in the area of human semen fractionation.
  • a method has been developed for separating semen populations into fractions enriched for healthy sperm using a bench top analytical-scale apparatus, termed the Microflow TM apparatus by Gradipore Limited. This apparatus was originally developed to separate macromolecules in small volumes and is described in WO 01/78878 .
  • Semen samples were obtained by masturbation through an internal donor program, with a minimum of three days abstinence. All samples were liquefied at RT for 30 min and subjected to routine semen analysis performed according to the WHO guidelines (WHO, 1999). All investigations involving the use of human materials were approved by the University of Newcastle Human Ethics Committee.
  • eosin dye exclusion test Human sperm samples were assessed for vitality using eosin dye exclusion test. This test relies on the fact that intact and functional plasma membranes of live cells are able to exclude extracellular substances, however, dead cells have non-functional or damaged plasma membranes, and subsequently cannot exclude the dye.
  • Ten ⁇ l of a 0.05% eosin dye solution in PBS was mixed with 10 ⁇ l of sample containing sperm population on a slide. On examination under phase contrast at 400X magnification, the cytoplasm of live cells appeared clear in colour, whereas that of dead cells appeared to be coloured pink or red. 200 cells were scored for each sample, and results were recorded as a percentage of live cells.
  • the concentrations of relevant sperm populations were calculated by making a 1:20 dilution with sperm dilution fluid containing formalin to render the sperm immotile, and 10 ⁇ l was placed into one chamber of an improved Neubauer haemocytometer. Samples were then allowed to settle by placing the haemocytometer inside a humid covered Petri dish for 5 minutes. All 25 large squares of the haemocytometer were counted and the resulting number was divided by the conversion factor of 5 to determine the concentration of sperm, in millions of cells/ml.
  • Fractionated sperm cells for each electrophoretic time-point were fixed in 4% para-formaldehyde in PBS at 4°C for >15 min. Cells were then washed twice in filtered PBS and resuspended in PBS to an appropriate concentration for consistent smear preparation. Treatments were smeared onto pre-prepared poly-l-lysine coated slides and allowed to dry at RT. Dried smears were then assayed using TUNEL methodology or stored at -20°C for future analysis.
  • TUNEL assay was performed using the in situ cell death detection kit (Fluorescein) from Boehringer Mannheim (Mannheim, Germany). Reagents were diluted in 1 X PBS as the use of undiluted reagents in our experience created high levels of background and non-specific staining which hampered the analysis for TUNEL positive cells. Un-diluted terminal deoxynucleotidyl transferase (TdT) from calf thymus (EC 2.7.7.31) in storage buffer (25 ⁇ l) was mixed with label solution (nucleotide mixture in reaction buffer) diluted 1:5 with PBS (225 ⁇ l) to provide 250 ⁇ l of TUNEL reaction mixture.
  • TdT un-diluted terminal deoxynucleotidyl transferase
  • a negative control consisting of nucleotide solution without TdT enzyme was included in all experiments. Ten ⁇ l of relevant TUNEL treatment mix was added to the spermatozoa in each well and slides were incubated for 1 hr at 37°C in a humid chamber depriving the slides of light and preventing evaporative loss.
  • Fractionated sperm cells for each electrophoretic time-point were diluted in PBS to appropriate concentration for consistent smear preparation. Treatments were smeared onto pre-prepared poly-l-lysine coated slides and allowed to dry at RT. Dried smears were fixed in 95% ethanol for 15 min at RT then stained by a modification of the Papanicolaou method, as described by Belsey et al 1980 (Belsey, M.A., Eliasson, R., Gallegos, A.J., Moghissi, K.S., Paulsen, C.A., Prasad, M.R.N. 1980) Laboratory Manual for the Examination of Human Semen and Semen-Cervical Mucus Interactions. Press concern, Singapore) or stored at -20°C for future analysis.
  • Smears were viewed using a Zeiss Axioplan 2 microscope under bright field illumination employing a 100X oil-immersion objective and a total magnification of 1250X.
  • sperm morphology The classification and evaluation of sperm morphology, along with the normal criteria for whole spermatozoa, was established according to Menkveld et al., 1990.
  • the entire spermatozoon (head, neck, midpiece and tail) was taken into consideration for evaluation along with any germinal epithelium or other cell types present.
  • Spermatozoa were classified into one of seven groups, normal (whole sperm), large, small, elongated, duplicated and amorphous heads, all with or without the presence of a cytoplasmic droplet and/or tail, neck and/or midpiece defect (Menkveld et al., 1990).
  • the seventh group consisted spermatozoa with a normal head with a tail and/or a neck and/or a midpiece defect and/or the presence of a cytoplasmic droplet (Menkveld et al., 1990).
  • Tail, neck and midpiece defects, loose heads, germinal epithelium and unknown cells were recorded separately and expressed per 100 spermatozoa (Menkveld et al., 1990).
  • a minimum of 100 spermatozoa were analysed with the aid of an eyepiece micrometre.
  • samples where incubated in BWW at 37°C in the presence of 5% CO 2 in air for a minimum of 3 hr to capacitate the cells.
  • Sperm were then treated with the calcium ionophore A23187 for 30 min at a final working concentration of 2.5 ⁇ M to induce acrosome reactions.
  • An equivalent dilution of DMSO was used as a vehicle control for each sperm population.
  • Samples were then subjected to HOS vitality tests prior to staining for the acrosome reaction. These were first centrifuged at 1000 x g to remove treatment media, resuspended in HOS, and incubated for 1 hr at 37°C. These samples were then centrifuged at 1000 x g for 5 minutes to remove the media.
  • hypogaea lectin (1 mg/ml of PBS) for 15 minutes in the dark at 4°C. Slides were washed with PBS, mounted in Mowiol, and visualised by fluorescence microscopy. At least 100 cells were scored for each sample, and results were expressed as percentage of live acrosome-reacted spermatozoa.
  • the cell separation system isolated a highly purified subpopulation of spermatozoa characterized by significantly better morphology, improved vitality, motility and enhanced functional competence, compared with the excluded population.
  • the selected population of spermatozoa exhibited significantly lower levels of DNA damage than their unselected counterparts.
  • this selection process was achieved in a fraction of the time taken to purify human spermatozoa using alternative selection procedures such the Percoll gradient centrifugation or 'swim-up' methods.
  • the separated populations of spermatozoa also expressed lower levels of detectable DNA damage (4.5 ⁇ 2%) as compared to the excluded populations (8.3 ⁇ 3.6%) after 5 min electrophoresis.
  • the TUNEL assay revealed a significant difference (P ⁇ 0.001) in labelled cells between separated and excluded sperm populations, however, there was no significant increase in TUNEL-labelled cells with time.
  • the percentages of detected DNA damage for separated and excluded fractions are shown in Figure 3 , which demonstrates significantly (p ⁇ 0.001) lower levels of DNA damage for all separated populations and no significant effect of time on either separated or excluded time-dependent populations.
  • the motility of spermatozoa from all electrophoretic time-points was also assessed to evaluate differences between separated and excluded populations and potential effects of the treatment regime employed.
  • Five minute electrophoretic separations resulted in a mean sperm motility of 68 ⁇ 6% 1.5 hr after separation. This data showed a relatively small difference in motility (78 ⁇ 5%) compared to untreated populations.
  • sperm motility had recovered to 70 ⁇ 5%, which was 94 ⁇ 10% of the value recorded in the untreated sperm populations.
  • Figure 8 shows the CASA motility parameters for separated and excluded sperm populations 3.5 hr post-treatment, and is again characterized by acceptable motilities for both separated and excluded sperm, and no significant difference from those observed for the corresponding excluded time-points. There was no appreciable difference between the results 1.5 and 3.5 hr post treatment, which demonstrated a retained capacity for the motility parameters studied for all populations subjected to sperm cell electrophoresis.
  • CASA analysis showed no significant effect of electrophoretic treatment time or time elapsed post-treatment (up to 3.5 hr) on the motility parameters studied for either separated or excluded populations. This indicated that although separated sperm populations had a greater number of motile cells, the motion parameters of the cells in both populations cannot be discriminated in terms of their forward progressive velocities, path velocities and their track speeds.
  • One of the key attributes of the gamete separation system is that it can isolate spermatozoa exhibiting low levels of DNA damage. Since DNA damage in spermatozoa is clinically correlated with poor fertility, low rates of fertilization, impaired embryonic development, abortion and childhood disease, this facility should be of value in treating infertile couples whose pathological profile is dominated by DNA damage in the male germ line.
  • the present invention uses a membrane-based electrophoresis system based on the Gradiflow TM technology developed by Gradipore Limited, designed to enrich for, or remove, a cell population(s) from a cell suspension mixture of cell types.
  • the technology incorporates a disposable membrane cartridge consisting of at least two membranes housed within the separation unit of the instrument.
  • An arrangement of ⁇ 1 separation membrane(s) between 2 restriction membranes forms ( n +1) chambers, where n is the number of separation membranes inserted.
  • Electrophoresis buffer is positioned or circulates on the outside of the restriction membranes and performs the dual function of setting the pH of the system and cooling the electrophoresis chamber. Application of an electric potential induces migration of the cells within the separation unit. By selecting an appropriate separation membrane pore size, isolation of cells by size fractionation can be achieved.
  • the present invention details the first membrane-based electrophoretic system applied to the separation/enrichment of sperm population(s) from semen containing at variety of sperm types.
  • the present membrane-based electrophoresis technology can separate sperm based on the combination of size and electrophoretic mobility.
  • Advantages associated with the present invention for sperm separation are that it is rapid and gentle, and preserves the in vivo status of the sperm. This is in contrast to antibody-based methods which require an enzyme or chemical process to remove the affinity ligand bound to the cell surface, and thus may alter the cell surface properties.

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Claims (24)

  1. Procédé permettant de séparer un type de spermatozoïde d'une population de spermatozoïdes par électrophorèse, comprenant les étapes consistant à :
    introduire un échantillon contenant des spermatozoïdes dans une chambre d'échantillon d'un appareil d'électrophorèse comprenant une première chambre d'électrolyte ; une deuxième chambre d'électrolyte ; une première chambre d'échantillon disposée entre la première chambre d'électrolyte et la deuxième chambre d'électrolyte ; une deuxième chambre d'échantillon disposée à côté de la première chambre d'échantillon ; une première barrière perméable aux ions disposée entre la première chambre d'échantillon et la deuxième chambre d'échantillon ; une deuxième barrière perméable aux ions disposée entre la première chambre d'électrolyte et la première chambre d'échantillon ; une troisième barrière perméable aux ions disposée entre la deuxième chambre d'échantillon et la deuxième chambre d'électrolyte ; et des électrodes disposées dans les première et deuxième chambres d'électrolyte ; et
    appliquer un potentiel électrique entre les électrodes amenant au moins un type de spermatozoïde dans la première ou la deuxième chambre d'échantillon à se déplacer à travers la première barrière perméable aux ions jusque dans l'autre parmi la première ou la deuxième chambre.
  2. Procédé selon la revendication 1, selon lequel le type de spermatozoïde présente une caractéristique souhaitée choisie dans le groupe constitué de la motilité, de la robustesse, du sexe, de la constitution génétique, de la normalité morphologique, du potentiel fécondant et d'une combinaison de ceux-ci.
  3. Procédé selon la revendication 2, selon lequel le type de spermatozoïde possède un potentiel fécondant.
  4. Procédé selon la revendication 1, selon lequel le type de spermatozoïde présente une caractéristique non souhaitée choisie dans le groupe constitué d'une motilité médiocre, d'une morphologie médiocre, de niveaux élevés d'endommagement de l'ADN et de niveaux élevés de génération d'espèces réactives de l'oxygène.
  5. Procédé selon l'une quelconque des revendications 1 à 4, selon lequel l'échantillon de spermatozoïdes contient au moins deux populations de spermatozoïdes.
  6. Procédé selon l'une quelconque des revendications 1 à 5, selon lequel le potentiel électrique est appliqué jusqu'à ce qu'au moins un type de spermatozoïde atteigne une pureté souhaitée.
  7. Procédé selon l'une quelconque des revendications 1 à 6, selon lequel une ou plusieurs barrières perméables aux ions sont des membranes d'électrophorèse présentant une taille de pore moyenne et une distribution de taille de pores caractéristiques.
  8. Procédé selon la revendication 7, selon lequel la totalité des barrières perméables aux ions sont des membranes présentant une taille de pore moyenne et une distribution de taille de pores caractéristiques.
  9. Procédé selon l'une quelconque des revendications 1 à 8, selon lequel la première barrière perméable aux ions est une membrane à pores de grande taille.
  10. Procédé selon la revendication 9, selon lequel la première barrière perméable aux ions est une membrane de polycarbonate présentant une taille de pore comprise entre environ 1 et environ 10 µm.
  11. Procédé selon l'une quelconque des revendications 1 à 10, selon lequel l'électrophorèse est réalisée avec une gamme de tension comprise entre 1 et 200 V.
  12. Procédé selon l'une quelconque des revendications 1 à 10, selon lequel l'électrophorèse est réalisée avec un courant appliqué allant jusqu'à environ 75 mA.
  13. Procédé selon l'une quelconque des revendications 1 à 10, selon lequel l'électrophorèse est réalisée avec une intensité de champ comprise entre 1 et 100 V/cm.
  14. Procédé selon la revendication 13, selon lequel l'intensité de champ est comprise entre 16 et 20 v/cm.
  15. Procédé selon l'une quelconque des revendications 1 à 14, selon lequel l'électrophorèse est réalisée pendant une durée allant de quelques secondes à environ 15 minutes dans un appareil présentant un volume d'échantillon compris entre environ 100 µl et environ 4 ml.
  16. Procédé selon l'une quelconque des revendications 1 à 15, selon lequel les spermatozoïdes sont dilués dans un tampon ayant une concentration comprise entre 1 et 100 mM.
  17. Procédé selon l'une quelconque des revendications 1 à 16, selon lequel des concentrations de spermatozoïdes comprises entre environ 0,1 et 250 × 106/ml sont traitées.
  18. Procédé selon la revendication 17, selon lequel des concentrations de spermatozoïdes comprises entre 15 x 106/ml et 140 x 106/ml sont traitées.
  19. Procédé selon l'une quelconque des revendications 1 à 18, selon lequel au moins environ 50 % du type de spermatozoïde restent viables ou sensiblement inchangés après la séparation.
  20. Procédé selon la revendication 19, selon lequel au moins environ 60 % du type de spermatozoïde restent viables ou sensiblement inchangés après la séparation.
  21. Procédé selon la revendication 20, selon lequel au moins environ 70 % du type de spermatozoïde restent viables ou sensiblement inchangés après la séparation.
  22. Procédé selon la revendication 21, selon lequel au moins environ 80 % du type de spermatozoïde restent viables ou sensiblement inchangés après la séparation.
  23. Procédé selon la revendication 22, selon lequel au moins environ 90 % du type de spermatozoïde restent viables ou sensiblement inchangés après la séparation.
  24. Utilisation d'un appareil d'électrophorèse à base de membranes pour séparer un type de spermatozoïde d'une population de spermatozoïdes.
EP04761402A 2003-10-07 2004-10-07 Separation de spermatozoides par electrophorese Expired - Lifetime EP1689853B1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
AU2003905474A AU2003905474A0 (en) 2003-10-07 Gamete Separation
PCT/AU2004/001367 WO2005033295A1 (fr) 2003-10-07 2004-10-07 Separation de spermatozoides par electrophorese

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EP1689853A1 EP1689853A1 (fr) 2006-08-16
EP1689853A4 EP1689853A4 (fr) 2007-07-11
EP1689853B1 true EP1689853B1 (fr) 2009-03-04

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EP2361967A1 (fr) * 2010-02-26 2011-08-31 Assistance Publique - Hôpitaux de Paris Procédé de séparation de gamètes, compositions et utilisations correspondantes
WO2013136091A1 (fr) 2012-03-16 2013-09-19 Fertility Innovations Limited Traitement des spermatozoïdes
GB201210496D0 (en) * 2012-06-13 2012-07-25 Fertility Innovations Ltd Method and apparatus for sperm enrichment
US9034161B2 (en) 2012-08-23 2015-05-19 Douglas T. Carrell Sperm separation devices and associated methods
US10379026B2 (en) 2012-08-29 2019-08-13 Inguran, Llc Cell processing using magnetic particles
DK2890498T3 (en) 2012-08-29 2018-05-22 Inguran Llc MAGNETIC REMOVAL OR IDENTIFICATION OF DAMAGED OR COMPRIMATED CELLS OR CELL STRUCTURES
EP2717034A1 (fr) * 2012-10-08 2014-04-09 Roumen Parapanov Procédés et kits pour l'évaluation de la qualité du sperme et utilisations de ceux-ci
WO2018071978A1 (fr) * 2016-10-20 2018-04-26 Memphasys Limited Séparation de sperme par électrophorèse
WO2018071977A1 (fr) * 2016-10-20 2018-04-26 Memphasys Limited Dispositif d'électrophorèse
US11578303B2 (en) 2017-01-31 2023-02-14 Genea Ip Holdings Pty Limited Method and system for processing a biological sample
CN110967230B (zh) * 2019-11-22 2022-05-31 珠海高瑞特医疗科技有限公司 一种精子活性氧含量的测定方法及试剂盒
EP3851853A1 (fr) * 2020-01-17 2021-07-21 Biotechnologicky Ustav AV CR, v.v.i. Procédé de séparation des spermatozoïdes dont la tête est intacte et non endommagée des spermatozoïdes dont la tête est endommagée et des cellules somatiques
CN111323361B (zh) * 2020-03-17 2022-05-10 南通大学 一种快速分离精子头、精子尾和正常有活力精子的方法

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AU2004278448B2 (en) 2007-06-07
DE602004019804D1 (de) 2009-04-16
WO2005033295A1 (fr) 2005-04-14
US20090101507A1 (en) 2009-04-23
ATE424449T1 (de) 2009-03-15
EP1689853A4 (fr) 2007-07-11
EP1689853A1 (fr) 2006-08-16
US8123924B2 (en) 2012-02-28
AU2004278448A1 (en) 2005-04-14

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